*2.1.1. Biopsy collection*

110 New Approaches to the Study of Marine Mammals

Ocean pollution is a threat to marine mammals. Some pollutants cause obvious and direct harm to the animals such as plastics and other debris. Others can be less evident, such as, agricultural runoff and industrial wastes. Industrial wastes can include air pollution that the animals breathe in and pollutants that can be found at different levels of the water column.

Whales are exposed to all environmental pollutants that reach the ocean. They spend time at the surface and travel throughout the water column so they are exposed to pollutants that remain at the surface and those that disperse through the water column. They experience dermal exposure through pollutants in the water. They feed at different depths so they get pollutants through ingesting animals that may have accumulated them. They all breathe air and, thus, are exposed to air pollutants. Consequently, they make excellent models to use for studying the threats and consequences of ocean pollution. The challenge, of course, is to develop an approach for studying the toxicology of marine pollution in marine mammals

that provides species specific data along with an individual and population context.

Marine mammal research is difficult and expensive. There are laws that have been implemented to specifically protect marine mammals. Some species not only fall under this protection but also are protected by the Endangered Species Act. Thus, research with these animals is strictly enforced and regulated through various permitting agencies (eg, National Marine Fisheries Service (NMFS) and United States Fish and Wildlife Service, USFWS). These permits limit the amount, time and ways they can be studied. In addition to the permits, properly trained personnel and proper equipment are required. Whales in particular are difficult to study because they are typically found far from shore requiring extensive travel and vessel time. There is specialized equipment to aid in finding the whales while they are underwater but most of the search requires visual sightings of animals for the short period of time they are at the surface, requiring trained crew to be on watch during all daytime hours in a variety of environmental conditions. Because of these factors, researching whales is a particularly challenging task and one that requires careful use of

A unique and effective way to study marine mammals is through skin biopsies. The skin can be used to determine the genetics of an individual which can allow for gender identification as well as determining intraspecific relationships, genetic diversity of different subpopulations and tracking individual animals over time. Biopsies can provide important information about environmental pollutants and their effects. For example, they can be used to measure the levels of metals because many metals are known to accumulate in the skin. Through a skin biopsy, blubber is also collected which can be measured for a variety of organic pollutants. In addition, the interface between the skin and blubber can be used to create living cell lines. These cell lines can further be used to determine the toxicity of a

In this chapter we use chromium as an example for how skin biopsies of free ranging whales can be used to evaluate the environmental impact of a particular ocean contaminant. We chose to use chromium because it is a known carcinogen and a known reproductive and

These types of pollutants can have long term and persistent exposures.

resources and the ability to extract as much information as possible.

pollutant by measuring levels of toxicity and DNA damage.

Whale skin biopsies were collected using a specialized biopsy dart and crossbow according to standard methods [4]. The biopsy dart has a stainless steel tip that collects a skin sample that is about 25 mm long or less and 7 mm in diameter. A buoyant stopper located behind the tip prevents the biopsy dart from penetrating beyond the depth of the tip. The stopper causes the dart to bounce off and float for an easy retrieval with a net. Tips are stored in 70% ethanol until use. Upon retrieval, the tissue sample is removed from the tip using Teflon forceps and placed into a glass Petri dish.

## *2.1.2. Biopsy processing*

The skin and blubber were separated leaving the interface to be used for skin fibroblast cell growth. The blubber and skin were used for analysis of genetics, levels of metals and organics. The interface, once isolated, was immersed in a tissue buffer (PBS with 20% penn/strep and 2% gentamicin) for 30 minutes to get rid of any bacteria that may have been present on the skin. Tissue was then placed in a Petri dish and cut into approximately 1 mm pieces. These pieces were transferred into two T-25 flask with 1 ml of medium (DMEM-F12, cosmic calf serum, L-glutamine, penicillin, streptomycin, sodium pyruvate) and placed upside down in a 33°C humidified incubator with 5% CO2. After 24 h, 5 ml of medium was added and the flask was gently turned right side up and monitored for cell growth. Living cells typically plated out on the flask directly from the tissue explants within one week.

## *2.1.3. Measuring Levels in the Skin from the Biopsy*

The whale skin biopsies were analyzed using inductively coupled plasma mass spectroscopy to determine the total chromium in the tissue according to published methods using a Perkin-Elmer/Sciex ELAN ICPMS. Samples were rinsed with deionized water and allowed to air dry in a laminar flow hood to minimize contamination. Approximately 0.1 g of tissue was placed in a digestion vessel, 2 ml of Optima grade nitric acid was added, the vessel placed in a hot block, and refluxed at 95°C for 4 h. The sample was cooled, 2 ml Optima grade hydrogen and deionized water (3:2 v/v) was added, heated until the effervescence subsided, cooled, and brought up to a final volume of 20 ml. Standard quality assurance procedures were employed (Table 1) and include the analysis of standard

reference materials, a duplicate sample and a pre-digestion spike. Instrument response was evaluated initially and after 10 samples, using commercially available calibration verification standards and a blank. All calibration verifications were within the acceptance criterion of 85-115% recovery and the preparation blank values were below 3x the limit of detection. Standard reference materials were used to assess method performance, where applicable. Interference check solutions were analyzed with all sample runs to check for matrix effects which might be interfering with sample analysis. The mean limit of detection (LOD) was the lowest analyte concentration likely to be reliably distinguished from the blank and at which detection is feasible. The LOD was previously determined by utilizing both the measured blank and test replicates of a matrix matched sample known to contain a low concentration of analyte. All samples were diluted 2x for analysis by ICP-MS. All data are presented as ug/g wet weight.

Skin Biopsy Applications in Free Ranging Marine Mammals: A Case Study of Whale Skin Biopsies

swell the cells, fixed and dropped onto slides. Slides were stained with Giemsa,

Once a skin biopsy was collected from a free ranging whale the skin was used to measure the levels of chromium present. The different valence states of chromium cannot accurately be determined, so we measured the total level of chromium in the tissue (Figure 1). For North Atlantic right whales in the Bay of Fundy, 7 biopsies were collected. The total chromium levels in right whale skin (wet weight) ranged from 4.9 to 10 ug Cr/g tissue with an average of 7.0 ug Cr/g tissue. (Figure 1A). For sperm whales, 331 biopsies were collected from 17 different regions around the globe. In sperm whales, the total chromium ranged from 0.9 to 122.6 ug Cr/g tissue with an average of 9.3 ug/g w.w (Figure 1B). The highest mean levels by region were found in the Bahamas with an average of 81.9 ug/g w.w for 2 animals. The average was slightly higher in sperm whale tissues than North Atlantic right whale tissues (Figure 1C).

coverslipped and 100 metaphases were analyzed per treatment concentration.

**3.2. Determining if Cr is cytotoxic and genotoxic to cultured whale cells** 

Both soluble and particulate forms of chromium are cytotoxic to sperm whale and Northern right whale skin cells. The data show that 1, 2.5, 5, 10 and 25 uM sodium chromate (the soluble form of chromate) induced 78, 60, 49, 11, 1 percent survival, respectively, in right whale cells; and 80, 51, 13, 2 and 0 percent survival, respectively, in sperm whale cells (Figure 2A). Doses of 0.1, 0.5, 1, 5 and 10 ug/cm2 lead chromate induced 126, 64, 49, 11 and 1 percent cell survival in right whale cells and doses of 0.05, 0.1, 0.5, 1, 5 and 10 ug/cm2 lead chromate induced 100, 91, 68, 63, 36 and 7 percent cell survival, respectively (Figure 2B).

Both soluble and particulate forms of chromium induced chromosome damage in sperm whale and North Atlantic right whale skin cells. We measured genotoxicity by induced chromosome damage in two ways: Percent of metaphases with damage and total damaged chromosomes in 100 metaphases. In sperm whale cells 1, 2.5, 5, 10 and 25 uM sodium chromate damaged 7, 17, 20 and 31 percent of metaphases, respectively, and induced 7, 16, 24 and 40 total aberrations, respectively. At the highest dose cell cycle arrest occurred and no metaphases were seen. In right whale cells 1, 2.5, 5 and 10 uM sodium chromate damaged 7, 15, 23 and 33 percent of metaphases, respectively, and induced 8, 18, 30 and 48 total aberrations, respectively (Figure 3A and 3B). For lead chromate, 0.5, 1, 3 and 5 ug/cm2 lead chromate damaged 6, 12, 15 and 27 percent of metaphases and induced 7, 13, 24 and 28 total aberrations, respectively in sperm whale cells. 0.5, 1, 2, 4 and 5 ug/cm2 lead chromate damaged 16, 19, 23, 32 and 26 percent of metaphases and induced 17, 22, 28, 40 and 30 total

**3.1. Determining if exposure has occurred** 

**3. Results** 

*3.2.1. Cytotoxicity* 

*3.2.2. Genotoxicity* 

aberrations, respectively (Figure 3C and 3D).

as a Valuable and Essential Tool for Studying Marine Mammal Toxicology and Conservation 113
